1. Field of the Invention
The present invention relates to the field of molecular biology. More specifically, the invention relates to methods and compositions for cleaving DNA molecules using transposases and preparing the cleaved DNA molecules for analysis by tagging the molecules with pre-selected sequences of interest. Among other things, the invention relates to the areas of in vitro amplification of nucleic acids, sequencing of nucleic acids, and screening of DNA libraries for sequences of interest.
2. Description of Related Art
Fragmentation of genomic DNA is a crucial step in DNA sample preparation for high-throughput sequencing. Traditionally used methods, such as DNA fragmentation using DNase I, are very unreliable and often result in DNA fragmentation that is either insufficient or too extensive. In either case, the yield of DNA fragments of useful size (about 200-800 base pairs (bp)) is low. This difficulty has been overcome by controlled DNA fragmentation using oligonucleotide-transposase complexes, such as the NEXTERA™ system from Epicentre. Such complexes are comprised of a dimer of modified Tn5 transposase and a pair of Tn-5 binding double-stranded DNA (dsDNA) oligonucleotides containing a 19 bp transposase-binding sequence, or inverted repeat sequence (IR). In the NEXTERA™ system, an engineered, non-native 19 bp transposase binding sequence is used, which provides more efficient DNA fragmentation than the native Tn5 IR sequence. This binding sequence is referred to as “mosaic”.
Unlike DNase, a single molecule of which can generate numerous breaks in a target DNA, the transposase complex is believed to create only one DNA cleavage per complex. Therefore, unlike with DNase I, the degree of DNA fragmentation is easily controlled during transposase fragmentation. Furthermore, specific nucleotide tags combined with the mosaic sequence can be attached in this transposase-mediated DNA fragmentation process, which is useful for DNA amplification in PCR and attaching the DNA fragments to sequencing chips.
Typically Mg(II) ions (also referred to herein as Mg+2 or Mg2+) are used as a cofactor with enzymes that exhibit DNA fragmentation activity. This is not surprising, as by examining the melting temperature of double-helical DNA in the presence of different metal ions it has been established that the preference for phosphate association was found to decrease in the order Mg(II)>Co(II)>Ni(II)>Mn(II)>Zn(II)>Cd(II)>Cu(II). The reaction of transition-metal complexes with polynucleotides generally fall into two categories: (i) those involving a redox reaction of the metal complex that mediates oxidation of the nucleic acid; and (ii) those involving coordination of the metal center to the sugar-phosphate backbone so as to mediate hydrolysis of the polymer. Though Mg(II) is the strongest binder, it is not necessarily the best cofactor. Enzymatic DNA cleavage can be influenced by many parameters, in particular by DNA conformation contingent upon the nature of the metal ion binding to the DNA. Furthermore, binding of a particular metal ion to the enzyme itself could affect protein conformation, protein-protein interactions, for instance protein dimerization that might influence the activity; and protein-nucleic acid interactions, i.e., modulating the speed of the reaction and the DNA cleavage specificity.
To date, the only transposase that is known to be suitable for DNA fragmentation and tagging is a modified Tn5 transposase. From the onset, Tn5 transposase has been problematic in several respects. First of all, the native transposase was practically impossible to produce, as it is toxic for E. coli when expressed from a strong promoter. However, it was possible to overcome this difficulty by deleting several N-terminal amino acids (Weinreich et al., J. Bacterial, 176: 5494-5504, 1994). Though this solved the toxicity problem, and the N-terminally truncated transposase was produced at high yield, it possessed very low activity. Therefore, several other mutations were introduced to increase its activity (U.S. Pat. No. 5,965,443; U.S. Pat. No. 6,406,896 B1; U.S. Pat. No. 7,608,434). However, this did not solve all of the problems. The mutated enzyme is stable only in high salt, such as 0.7M NaCl, (Steiniger et al., Nucl. Acids Res., 34: 2820-2832, 2006), but it quickly loses its activity at the low salt conditions that are required for the transposase reaction, with a half-life only 2.4 min in the reaction mixture. Thus, DNA fragmentation reactions using this transposase are typically performed in 5 minutes, and very large amounts of enzyme are used. Despite the fact that high salt concentration is maintained throughout the purification process, the purified enzyme is largely inactive; thus, 9.4 times excess of enzyme over nucleotides is typically used to form Tn5 transposase-oligonucleotide complexes (Naumann and Reznikoff, J. Bioi. Chem., 277:17623-17629, 2002). In addition, the transposase is prone to proteolytic degradation. Therefore, the degradation-prone sites were mutated. Interestingly, these mutations resulted in drastic reduction of the in vivo activity, but had little effect on the in vitro activity (Twining et al., J. Bioi. Chem., 276: 23135-23143, 2001). Overall, Tn5 transposase is difficult to produce, it is required in large amounts, and it is very expensive.